The invention relates to a liposomal formulation that is capable of fusing with cells.
Encapsulation of bioactive compounds in natural or synthetic matrixes has been extensively studied over the past decades. Advantages of encapsulation are numerous. First, it provides protection from the inactivation or degradation of the bioactive compound. Secondly, it controls the kinetics of compound release, allowing the optimization of the blood concentration profile. This optimization diminishes the deleterious effects of bioactive compounds with short half lives. In addition, it permits a reduction in toxicity, where relevant.
Liposomes are closed microscopic vesicles that form spontaneously from phospholipids above their transition temperature, in the presence of excess water. Vesicles with diameters ranging from 20 nanometers to several micrometers can be prepared. Multilamellar liposomes are made of concentric phospholipid bilayers separated by aqueous layers. Unilamellar liposomes consist of a single phospholipid bilayer surrounding an aqueous core. Liposomes can accommodate hydrophilic molecules in the aqueous spaces and lipophilic molecules in the lipid bilayers.
The potential of liposomes as vehicles for antimicrobial agents, or therapeutic liposomal formulations, has been studied by several investigators. Successful treatments with liposomes against intracellular bacteria have been demonstrated (Lopez-Berestein et al., 1987, U.S. Pat. No. 4,981,692). A number of studies have also shown that liposome-entrapped antibacterial agents increase the therapeutic indices of these agents as a result of decreased toxicity, modification of pharmacokinetics and tissue distribution parameters (Lagacxc3xa9 et al., 1991, J. Microencapsulation 8:53-61 and references therein; Omri et al., 1994, Antimicrob. Agents Chemother. 38:1090-1095)
Microorganism resistance to antibiotics is an important health problem world-wide. According to estimates from the United States Center for Disease Control and Prevention, for the period 1980 to 1992, approximately two million hospital-acquired infections occurred annually in the United States, accounting for more than eight million days extended hospital stay and generating more that $4 billion in additional health care costs each year. While overall per capita mortality rates for all diseases declined in the United States from 1980 to 1992, the per capita mortality rate due to infectious disease increased 58% over this period, making infectious diseases the third leading cause of death in the United States. Microbial infections, especially infections caused by difficult-to-treat, antibiotic-resistant microbes, such as bacteria and fungi, cause or contribute to a substantial majority of these deaths.
Microbes use different mechanisms to resist antibiotics. These mechanisms include prevention of the penetration, and/or extrusion, of the drugs from the microbial cells, enzymatic inactivation of the drugs, or alteration of the molecular target. Frequently, multiple mechanisms are present in a synergistic way, thus increasing the degree of resistance. Increasing evidence suggests that acquired antibiotic resistance is often due to a balance between outer membrane penetration rate and the subsequent enzyme inactivation rate. Thus, the outer membrane barrier and the antibiotic-degrading enzymes are strongly synergistic. New generation antibiotics, which can overcome strain-based enzymatic degradation, still do not solve the significant hurdle of penetration through the impermeable microbial membrane or through an exopolysaccharide layer of the microorganism and to its site of action. Recent research reports have also indicated that membranes with a low level of permeability, combined with a multiple drug efflux, play a dominant role in many antibiotic-resistant microorganisms, including Pseudomonas aeruginosa (Poole, K. et al., 1996, Antimicrob. Agents Chemother. 40:2021-2028).
The problem of increased resistance to antibiotics is compounded by the misuse of these agents (Merck manual, 1992, 16th Edition, Merck Res. Lab.). For example, because of the antibiotic resistance of microorganisms, which is more acute with older types of antibiotics, practitioners are often prompted to use a newer generation antibiotic, contributing to the increased resistance of microorganisms to these antibiotics. The large scale use of antibiotics in animals, including but not limited to dairy cows, and the presence of these antibiotics in milk, or in the environment, is yet another contributor to increases in microbial resistance to antibiotics.
Although antibiotics are useful for treating infections, their use can be accompanied by concentration-dependent toxicity and side effects. It is, therefore, important to ensure that their plasma concentrations do not exceed toxic levels. It is equally important to ensure that fear of toxicity does not result in a therapeutically inadequate dosage.
The encapsulation of antibiotics into liposomal formulations has been described (Lagacxc3xa9 et al., 1991, J. Microencapsulation 8:53-61; Boswell et al., J. Clinical Pharmacology 38:583-592 and references therein; Da Cruz et al., 1993, WO 93/23015 and Proffitt et al., 1994, WO 94/12155). Nevertheless, these formulations fail to display a very drastic enhancement of the therapeutic activity of the antibiotic as compared to its activity in the free form. Indeed, the preferred aminoglycoside (netilmicin) liposomal formulation of Da Cruz et al., which comprises phosphatidylcholine (PC), cholesterol and phosphatidylinositol (PI), only shows a modest increase activity in vivo with the aminoglycoside as part of the liposomal formulation as compared to free aminoglycoside (at best by a factor of three). Proffitt et al., disclose a different aminoglycoside (amikacin) liposomal formulation comprising PC, cholesterol and distearoyl phosphatidylglycerol (DSPG). Although the Proffitt et al., formulation appears to be superior at enhancing the in vivo therapeutic activity of the aminoglycoside as compared to that of Da Cruz, this increase is still relatively low and dependent on the tissue (10-fold in lung).
In view of the therapeutic, diagnostic, and research benefits incurring therefrom, it would be useful to have liposomes capable of fusing with pathogenic microbes and other cells.
We have now discovered that a liposomal formulation, previously known only to fuse with bacteria to deliver antimicrobials, is capable of delivering virtually any agent to a microbial cell, including a non-bacterial cell, or a macrophage. Accordingly, the invention provides compositions and methods for liposomal delivery of compounds to cells.
In the first aspect, the invention features a low-rigidity liposomal formulation, which is characterized as being free of cholesterol, and including neutral and anionic phospholipids at a molar ratio of 5:1 to 20:1, having a phase transition temperature (Tc) below 42xc2x0 C. as measured by differential scanning calorimetry (DSC), where said Tc is below about 42xc2x0 C., such that the formulation enhances fusion of the neutral and anionic phospholipids with a cell, where the formulation either does not include an antibacterial compound or where the formulation does not enhance penetration inside a bacterial cell.
In preferred embodiments of the first aspect, the formulation further comprises an agent, preferably an antimicrobial agent. In other preferred embodiments the cell is a macrophage or a non-bacterial microbial cell, preferably a fungus (e.g. a yeast), or the agent to be delivered is not an antimicrobial agent, for example, a nucleic acid encoding a commercially useful protein.
In other embodiments of the invention, the neutral and anionic phospholipids are present at ratios of about 8:1 to 18:1 or 10:1 to 15:1. The preferred neutral phospholipid may be dipalmitoylphosphatidylcholine (DPPC) or 1,2-di-o-hexadecyl-sn-glycero-3-phosphocholine (DHPC) and the preferred anionic phospholipid may be dimirystoylphosphatidylglycerol (DMPG) or soybean 1-xcex1-phosphatidylinositol (PI).
In other embodiments of the invention, the formulation may comprise two or more agents with different mechanisms of actions. In other embodiments of the invention, the agent may be an antibiotic, such as tobramycin or amphotericin B; a fungicide (preferably a fungicide that is not an antibacterial compound); a detergent; a nucleic acid, such as an antisense oligonucleotide or a nucleic acid encoding a cytotoxin; or a compound, such as a dye. The concentration of tobramycin or amphotericin B may range from 0.1 ug/ml to 500 mg/ml, preferably from 1 ug/ml to 50 mg/ml.
A second aspect of the invention provides a method of killing non-bacterial microbes. In preferred embodiments, the method includes treating, prevention, or diagnosis of a non-bacterial microbial infection in a mammal, comprising administration of a pharmaceutically effective amount of the liposomal formulation of the invention to the mammal. A related aspect of the invention provides a method of killing a microbe ex vivo, comprising administration of a pharmaceutically effective amount of the liposomal formulation of the invention to the microbe. In a preferred embodiment of this aspect, the microbe is in a cell culture medium.
A third aspect of the invention provides a method of treating a mammal, comprising administration of a pharmaceutically effective amount of the liposomal formulation to the mammal. The invention also provides a method of preventing a microbial infection, for example, a bacterial infection, in a mammal, comprising comprising administration of a pharmaceutically effective amount of the liposomal formulation of the invention to the mammal. In an embodiment of this aspect, the bacteria may be Pseudomonas aeruginosa, Burkholderia cepacia, Escherichia coli, and Staphylococcus aureus, and the mammal may be a human with cystic fibrosis or chronic infection. In another embodiment of this aspect, the formulation enhances penetration, by direct interaction with a microbe, of the agent through at least one of the microbial outer membrane and exopolysaccharide layer. Hence, the liposomal formulation of the present invention may provide increased efficacy in the prevention of mucoid bacterial infection, as is the case with, for example, bovine mastitis.
A fourth aspect of the invention provides method of diagnosing a microbial infection in a mammal, comprising administration of a pharmaceutically effective amount of the liposomal formulation, and an agent, to the mammal. In a preferred embodiment of this aspect, the agent is a dye.
A fifth aspect of the invention provides a method of delivering an agent inside a macrophage, comprising administration of the liposomal formulation of the invention to the macrophage.
A sixth aspect of the invention provides a method of delivering an agent inside a cell, comprising administration of the liposomal formulation of the invention to the cell.
In various embodiments of the above aspects, the mammal is a human and the administration is systemic, for example, for the treatment of septicemia. In an embodiment of these aspects, the infection is caused by at least one type of fungus, preferably a yeast, such as Candida, Histoplasma, Blastomyces, Coccidioides, Aspergillus, Mucomycosis, Microsporum, Epidermophyton, Trichophyton, and Cryptococcus species. In another embodiment, the infection is a respiratory infection, i.e. in an immunocompromised patient, and the yeast is Candida or Aspergillus fumigatus. In other embodiments, the microbial infection is Pneumocystis carinii pneumonia (PCP) and the formulation further includes pentamidine, a drug which is used for the prevention and treatment of PCP.
A seventh aspect of the invention provides a method of treating a drug-resistant microbial infection in a mammal, comprising administration of a pharmaceutically effective amount of the liposomal formulation of the invention to the mammal. In preferred embodiments of this aspect of the invention, the drug-resistant microbial infection is a bacterial infection or a yeast infection.
An eighth aspect of the invention provides a method of preventing proliferation of a drug-resistant microbe in a mammal, comprising the administration of a pharmaceutically effective amount of the liposomal formulation to the mammal.
A ninth aspect of the invention provides a use of the liposomal formulation for the treatment or prevention of a microbial infection in a mammal.
A tenth aspect of the invention provides a use of the liposomal formulation for the manufacture of a medicament for treating or preventing a microbial infection in a mammal.
An eleventh aspect of the invention provides a method of making the liposomal formulation, comprising mixing the neutral and anionic phospholipids in a solution; evaporating the solution to form a lipid film; hydrating the lipid film; extruding the hydrated lipid film through a suitable porous material; where the method lacks a lyophilization step.
By xe2x80x9cfree of cholesterolxe2x80x9d is meant a formulation that is lacking cholesterol sufficient to alter the stability of the liposomal formulation such that the Tc is increased by greater than 5% compared to a formulation without any cholesterol.
By xe2x80x9cphase transition temperaturexe2x80x9d or xe2x80x9cTcxe2x80x9d is meant the temperature at which liposomes destabilize or go into liquid phase, as measured by differential scanning calorimetry (DSC) or nuclear magnetic resonance (NMR). For the purposes of this invention, the Tc of the liposomes of the invention is destabilization above 35xc2x0 C. and liquid phase below 40xc2x0 C. as measured by NMR, or destabilization above 29xc2x0 C. and liquid phase below 42xc2x0 C., as measured by DSC. Preferably, the Tc, as measured by destabilization by DSC, is about 37xc2x0 C. or below the body temperature of the animal to be treated.
By xe2x80x9cenhances fusionxe2x80x9d is meant any increase in fusion of the liposomes of the invention with a cell when compared with liposomes that have a significant amount of cholesterol or other stabilizing agent.
By xe2x80x9cenhance penetrationxe2x80x9d is meant any increase in the penetration or delivery of an agent inside a cell by enhanced fusion of the cell with the liposomes of the invention.
By xe2x80x9cagentxe2x80x9d is meant any compound or chemical, be it naturally-occurring or artificially-derived. The term agent is designed to include, but is not limited to antibiotics, fungicides, detergents, bioactive molecules, such as proteins or parts thereof, nucleic acids or part thereof, amino acid analogs or nucleoside analogs, contrast and diagnostic materials (e.g. dyes), cytotoxins, growth factors, hormones, such as corticosteroids, or components thereof. The term agent includes a combination of more than one agent.
By xe2x80x9cantimicrobial agentxe2x80x9d is meant any agent that is capable of causing death or preventing proliferation of a microbe.
By xe2x80x9cdrug-resistantxe2x80x9d is meant any microbe that is capable of surviving what was previously determined to be lethal concentrations of an agent.
By xe2x80x9cpharmaceutically effective amountxe2x80x9d is meant an amount of an agent sufficient to produce a healing, curative, or ameliorative effect in the treatment or prevention of a microbial infection.
Other features and advantages of the invention will be apparent from the detailed description of the preferred embodiments given hereinafter. However, it should be understood that the detailed description, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art.